Expression of RNA polymerase I catalytic core is influenced by RPA12

RNA Polymerase I (Pol I) has recently been recognized as a cancer therapeutic target. The activity of this enzyme is essential for ribosome biogenesis and is universally activated in cancers. The enzymatic activity of this multi-subunit complex resides in its catalytic core composed of RPA194, RPA135, and RPA12, a subunit with functions in RNA cleavage, transcription initiation and elongation. Here we explore whether RPA12 influences the regulation of RPA194 in human cancer cells. We use a specific small-molecule Pol I inhibitor BMH-21 that inhibits transcription initiation, elongation and ultimately activates the degradation of Pol I catalytic subunit RPA194. We show that silencing RPA12 causes alterations in the expression and localization of Pol I subunits RPA194 and RPA135. Furthermore, we find that despite these alterations not only does the Pol I core complex between RPA194 and RPA135 remain intact upon RPA12 knockdown, but the transcription of Pol I and its engagement with chromatin remain unaffected. The BMH-21-mediated degradation of RPA194 was independent of RPA12 suggesting that RPA12 affects the basal expression, but not the drug-inducible turnover of RPA194. These studies add to knowledge defining regulatory factors for the expression of this Pol I catalytic subunit.


Introduction
RNA Polymerase I (Pol I) is a multi-subunit enzyme that operates in the nucleolus of the cell [1][2][3]. The key enzymatic activity of Pol I is to transcribe the ribosomal DNA (rDNA) into the 13 kb long 47S precursor ribosomal RNA (rRNA), which is the first step of the complex, resource and energy-consuming process of ribosome biogenesis [1,2,4]. Ribosome biogenesis is especially critical at times requiring extensive protein synthesis, such as cellular divisions during development and regeneration or for highly specialized functions within differentiated cells [5][6][7]. In this process transcription by Pol I is the rate limiting factor [1,2,8] Colis et al. and verified for purity using liquid chromatography/ mass spectrometry and 1H nuclear magnetic resonance [13]. Yeast cells were grown at 23˚C on Yeast Peptone Dextrose (YEPD) agar plates supplemented with indicated concentrations of BMH-21.

Immunoprecipitation
Cells were harvested in RIPA lysis buffer supplemented with protease inhibitors (Roche). Protein lysates (1 mg) were precleared using Dynabeads Protein G beads (Invitrogen 10003D) for 1 hour at 4˚C and centrifuged at 5,000 rpm for 5 minutes at 4˚C. Primary antibodies (2 μg) against RPA194 (C-1; Santa Cruz Biotechnology), RPA135 (H-15; Santa Cruz Biotechnology) or control anti-mouse IgG (Millipore Sigma) were incubated with the supernatant overnight with rotation at 4˚C. The protein-antibody mixture was collected on Dynabeads Protein G beads (50 μL per sample) for 45 minutes at 4˚C followed by 5 washes with RIPA buffer with protease inhibitor (Roche). The beads were resuspended in 2x Laemmli Sample Buffer with DTT and boiled for 10 minutes. Samples were run on SDS-PAGE gel and transferred to PVDF membranes (Millipore Sigma) followed by immunoblotting.

Chromatin immunoprecipitation
Cells were fixed with 1.1% formaldehyde for 6 minutes, washed with PBS, scraped, normalized according to cell numbers, and pelleted at 4˚C using 500 x g. Lysis was completed using the iDeal ChIPseq kit (Diagenode, Cat# C01010170). Following chromatin isolation, chromatin was resuspended in iS1b buffer (Diagenode) and sheared using Covaris ME220 Focused-ultrasonicator. Immunoprecipitation was conducted using POLR1A/RPA194 (C-1; Santa Cruz Biotechnology) (5 μg) antibodies for 6 hours and the precipitates were collected on Dynabeads G beads (Thermo Fisher) at 4˚C. The beads were washed, and the chromatin was eluted and purified. qPCR was conducted as above.

Statistical analysis
The following statistical methods were conducted using a minimum of three independent biological replicates: Analysis of variance (ANOVA), Student's two-tailed t test and non-parametric Mann-Whitney two-tailed t tests, using Excel or GraphPad Prism software. The test used for each experiment is indicated in the figure legend. The p values were expressed as follows: ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; and ****, p<0.0001.

Effect of Pol I inhibitor BMH-21 on RPA12
RPA12, the small 13.9 kDa subunit specific for Pol I, is required for the cleavage of nascent rRNA and assists in polymerase backtracking and proofreading [20,26,27]. Based on detailed dynamic studies in vitro, RPA12.2, the yeast RPA12 homologue, affects nucleotide addition kinetics and elongation complex stability [19,20]. We have earlier shown that BMH-21, a small-molecule Pol I inhibitor, destabilizes RPA194 [12]. However, the effect of BMH-21 on RPA12 has not been analyzed before. We first conducted a kinetic experiment to assess the impact of BMH-21 on RPA12 using immunofluorescence analysis. BMH-21 (1 μM) was applied to the cells for increasing periods of time, up to 180 minutes, followed by staining of the cells for RPA12 ( Fig 1A). Pol I transcription stress by BMH-21 leads to nucleolar reorganization, is amply documented [12,39], and was monitored by staining for fibrillarin (FBL), an RNA methyltransferase and dense fibrillar center protein required for rRNA processing. BMH-21 treatment caused rearrangement and condensation of the nucleolus at 30-60 minutes of treatment. Nucleolar caps became prominent at 180 min. RPA12 was observed on the outer surface of the nucleolar caps at this time, and partially overlapped with FBL that resided innermost in the caps (Fig 1A). These findings are consistent with the location of Pol I complex proteins during transcription stress [39]. A reduction in RPA12 signal was observed at 180 minutes (Fig 1B), indicating either a decrease in its protein expression or its subcellular redistribution. As we experienced a limitation in the RPA12 antibody performance during immunoblotting analysis (S1 Fig), we ectopically expressed RPA12 tagged with DDK and treated the cells with BMH-21. We observed a decrease in RPA12-DKK supporting the notion that RPA12 abundance could be affected by the drug treatment (Fig 1C and 1D). BMH-21 treatment of the cells did not affect the expression of RPA12 transcript (Fig 1E).

Influence of RPA12 depletion on Pol I subunits
To investigate the roles of RPA12 in mammalian models, we sought to determine the impact and function of RPA12 in human cells using RNAi against RPA12 using stable lentiviral

PLOS ONE
shRNA knockdown. However, we did not achieve stable, substantive knockdown of RPA12 over serial passaging using RPA12 shRNAs despite the acute knockdown being effective (S2 Fig). Therefore, transient siRNA treatment using multiple siRNAs against RPA12 was used for cellular knockdown and was validated using qPCR (Fig 2A). An 80% reduction in cellular RPA12 expression was achieved, with little variance between biological replicates. We additionally used immunofluorescence analysis to detect RPA12 protein levels, which showed prominent decrease in RPA12 protein expression (Fig 2B and 2C).
We then analyzed the impact of RPA12 knockdown on Pol I subunits using immunofluorescence. Compared to control siRNA-transfected cells (siCtrl), RPA12 knockdown cells displayed a decrease in the signal intensity of RPA194, the largest catalytic subunit of Pol I (Fig  2D and 2E). RPA135, the second largest subunit, showed a shift in localization from the nucleolus to nucleoplasm upon RPA12 knockdown (Fig 2D). Similar changes were not observed for FBL (Fig 2D) or the other Pol I subunits PAF53 and CAST (Fig 2F and 2G). These findings imply RPA12 depletion impacts the enzyme complex in a Pol I subunit-specific manner that does not activate a nucleolar stress response.

RPA12 and BMH-21 affect the expression and stability of Pol I subunit RPA194
Next, we asked whether the Pol I inhibitor-induced destabilization of RPA194 is dependent on RPA12. We treated control siRNA-transfected and RPA12 knockdown cells with BMH-21, followed by immunofluorescence staining for RPA194 and FBL as control ( Fig 3A). As in Fig 2D  and 2E, the signal for RPA194 decreased in the RPA12 knockdown cells (Fig 3B). As previously documented [3,12], BMH-21 treatment led to a robust decrease in RPA194 nucleolar staining in both the control and RPA12 depleted cells (Fig 3A and 3B).
We then analyzed whether RPA12 affects RPA194 steady-state level by western blotting. As shown by a representative experiment, and quantification of multiple biological replicates, RPA12 knockdown led to a significant 30% decrease in RPA194 protein expression (Fig 4A  and 4B). However, there was no change in RPA135 protein by RPA12 knockdown (Fig 4A and  4B). Further assessment into the effect of either RPA12 knockdown, treatment with BMH-21, or their combination on the transcripts of RPA194 or RPA135 by qPCR did not reveal any change, indicating that the changes observed in RPA194 protein are likely posttranscriptional (S3A and S3B Fig).
We next analyzed the dependency of RPA194 turnover by BMH-21 on RPA12. Consistent with our previous publication [12], BMH-21 caused a prominent decrease of RPA194 in cells transfected with control siRNA and this decrease was not significantly altered by the knockdown of RPA12 (Fig 4A and 4B). We observed a decrease in RPA135 by the drug treatment; however, RPA12 knockdown did not modify this response (Fig 4A and 4B). Thus, we conclude that while RPA12 affects the basal stability of RPA194, the drug-induced turnover of RPA194 is independent of RPA12 expression.
In the yeast, A190 (corresponding to human RPA194) and A135 (corresponding to human RPA135) form a stable stochiometric complex [40]. Since we observed that RPA12 knockdown affected RPA194 protein expression and RPA135 localization we questioned whether these alterations could cause complex dissociation and the loss of enzymatic activity upon RPA12 knockdown. We therefore performed a Co-IP experiment to determine the effect of RPA12 on their interaction and used either RPA194 or RPA135 antibodies for the pulldowns followed by reciprocal immunoblotting for RPA194 and RPA135 (Fig 4C and 4D). The co-precipitation of either RPA194 or RPA135 was independent of RPA12 knockdown (Fig 4D). These findings indicate that the RPA194:RPA135 complex does not dissociate upon RPA12 knockdown.

RPA12 depletion does not affect Pol I transcription activity or Pol I occupancy on rDNA
To further investigate whether Pol I activity is impacted by knockdown of RPA12 we analyzed the expression of the 47S precursor rRNA transcript by qPCR using primers specific to the short-lived 5'ETS rRNA and the stable mature 18S rRNA (Fig 5A). RPA12 knockdown did not affect the expression of the 5'ETS or 18S rRNAs. As expected, BMH-21 treatment robustly

PLOS ONE
decreased expression of the 5'ETS rRNA, while it had no effect on the 18S rRNA ( Fig 5A). RPA12 knockdown did not alter these drug-induced responses.
We wanted to further assess this outcome using Northern blotting. We used an internal transcribed spacer 1 (ITS1) probe specific to the precursor rRNA and a 28S probe specific to the mature rRNA, which also served as loading control (Fig 5B). First, we evaluated the temporal response to BMH-21. We observed a rapid reduction of the 47S precursor rRNA within 15 minutes, and a complete loss of the 21S, 26S, 34S, 41S rRNA processing intermediates by 6 hours (Figs 5C and S4A). Then we evaluated the effect of RPA12 knockdown on the rRNA synthesis and included as a control the knockdown of RPA135 (S4B and S4C Fig). While RPA135 knockdown prominently abrogated rRNA synthesis, RPA12 knockdown did not affect the processing intermediates and had only a minor effect on the 47S precursor rRNA (Figs 5D and S4D). BMH-21 abrogated rRNA synthesis in both the RPA12 and RPA135 knockdown cells (Fig 5D).
Lastly, we conducted chromatin immunoprecipitation (ChIP) analysis to assess whether RPA12 affects Pol I occupancy on the rRNA gene. ChIP was conducted on RPA12 knockdown and siCtrl cells using primers for the coding region (promoter, 5'ETS, 18S), two termination sites (T1, T2) and the non-coding intragenic spacer (IGS). There was no major difference in the occupancy of RPA194 on the rRNA gene locus upon RPA12 knockdown (Fig 5E). We previously demonstrated that the effects of BMH-21 on rDNA transcription are conserved across eukaryotic species, including yeast [14,15,17]. To determine whether complete deletion of RPA12.2 influences cellular responses to BMH-21, we plated WT, rpa12.2Δ, and dst1Δ cells on YEPD and grew cells for six days at 23˚C (Fig 6). DST1 encodes the transcription factor TFIIS. Both RPA12.2 and TFIIS influence nascent RNA cleavage and serve similar roles for Pols I and II respectively. Thus, dst1Δ was included as a negative control.
Interestingly, rpa12.2Δ cells formed colonies even in the presence of 30 μM BMH-21, whereas WT and dst1Δ cells were less viable in the presence of BMH-21 and colonies that did form were smaller, indicating slower cell proliferation (Fig 6). These data show that the presence of RPA12 renders Pol I more sensitive to inhibition by BMH-21. The simplest interpretation of this observation is that Pol I forms a less stable transcription elongation complex when RPA12 is expressed [20]. Deletion of RPA12.2 results in more stable transcription elongation complexes and reduced transcription elongation rate by Pol I. These properties may render Pol I more resistant to the transcriptional stress induced by BMH-21, much like Pol II.

Discussion
RPA12 is bestowed with several key activities in Pol I transcription. These include nucleotide addition, RNA cleavage, enzyme backtracking and proofreading, and transcription termination [18,20,26,27,41]. Not surprisingly, RPA12.2 deletion has temperature-associated  qPCR analysis of rRNA synthesis following transfection with siCtrl and RPA12 siRNAs in A375 melanoma cells. Cells were treated with and without BMH-21 (1 μM) for 6 hours. Primer pairs for 5'ETS and 18S rRNAs were used. Fold change for n = 3 biological replicates are shown, and data are represented as mean ± SD. Statistical analysis was conducted using nonparametric Mann-Whitney two-tailed test. ns, non-significant. (B) Schematic outline for the rRNA coding region and the probes used for Northern analysis (blue) and primers used for ChIP (red). (C and D) Northern blot analyses. (C) A375 cells were treated with BMH-21 for the indicated times and total RNA was prepared. 6 μg RNA/lane was loaded. (D) Total RNA was isolated from A375 melanoma cells transfected with siCtrl, RPA12 or RPA135 siRNAs and treated with or without BMH-21 for 6 hours. RNA (0.9 μg per lane) was loaded and the blots were probed with ITS1 and 28S probes as indicated. rRNA processing transcripts are indicated to the left. (E) Chromatin immunoprecipitation analysis following siCtrl and RPA12 knockdown in A375 melanoma cells. Primer pairs for the promoter (-48), 5'ETS (+851), 18S (+4446), two termination sites (+13508, +15364) and non-coding IGS (+30541) regions were used. Fold enrichment of n = 4 biological replicates is shown. Data are represented as mean ± SD. Statistical analysis was conducted using non-parametric Mann-Whitney two-tailed test. ns, non-significant. lethality in yeast. However, when grown at cold temperatures, or if only the C-terminus containing the TFIIS paralog domain is deleted, viability is sustained [24,42]. We were unsuccessful in establishing long-term stable knockout of RPA12 using shRNA or sgRNAs (not shown) in mammalian cancer cells, while transient knockdown was achieved using both shRNA and siRNA. This suggests that RPA12 knockdown does not immediately compromise mammalian cell proliferation; however, the sustained, long-term depletion of RPA12 leads to loss of cell viability. Further, we show that knockdown of RPA12 in mammalian cells leads to decreased expression of Pol I catalytic subunit RPA194. Despite this, Pol I gene occupancy, transcription, and rRNA synthesis remain unaffected in these short-term experiments. We infer that cancer cells tolerate transient fluctuations in the expression of their large subunits and maintain stable association with the gene throughout transcription cycles possibly due to their abundant expression.
The small-molecule Pol I inhibitor BMH-21 targets Pol I via intercalation of the GC-rich rDNA leading to inhibition of transcription initiation and elongation, ultimately leading to the degradation of Pol I subunit RPA194 [12,[14][15][16][17]. Given that the elongation blocks require enzyme backtracking and RNA cleavage for resolution we analyzed the effect of this Pol I inhibitor on RPA12. Following application of the inhibitor onto cells, RPA12 was detected within an hour in nucleolar cap structures and over time its expression in the nucleolus was reduced. Our data show a reduction in ectopically expressed RPA12 suggesting that also its turnover could be affected by BMH-21. However, we are unable to confirm this finding on the endogenous protein due to the limitation in the performance of the antibody in western blotting analyses. This prompted our investigation into whether RPA12 knockdown influences the stability of Pol I subunits, either at steady-state or in response to Pol I inhibitor, and their ability to form a stable core complex.
We analyzed the expression of Pol I subunits, specifically the two largest subunits RPA194 and RPA135 following depletion of RPA12. We observed a decrease in RPA194 protein and a shift of RPA135 from the nucleolus into the nucleoplasm. The decrease in RPA194 was specific to this subunit, since RPA12 knockdown had negligible impact on RPA135 steady-state level. This is interesting as the stoichiometric balance of human Pol I shown by structural reconstructions denote one RPA194 subunit to one RPA135 subunit [43]. Hence it is possible that instability or delocalization of one subunit could disrupt the binding or structure of Pol I. However, Co-IP studies indicate that despite the decrease in RPA194 expression and relocalization of RPA135 there is no dissociation of the core complex formed by RPA194 and RPA135 upon RPA12 knockdown. The findings reported here are consistent with those in yeast as Nogi et al. found that A190, the RPA194 yeast homologue, had decreased expression in RPA12.2 deletion strain [42] and that RPA12 is involved in the recruitment and docking of the catalytic core to the Pol I complex [18,24,42]. Thus, our data supports these findings by indicating a role for RPA12 in human Pol I stability.
Even if the structure and core complex of Pol I is not disrupted by RPA12 knockdown we postulated RPA12 knockdown could cause functional changes that negatively affect the transcriptional capacity of Pol I. To determine whether RPA12 knockdown has an impact on Pol I transcription, RNA was isolated from RPA12 knockdown cells and the mature and intermediate transcriptional products were analyzed. There was no major change in Pol I transcription following RPA12 knockdown of the short-lived 5' ETS transcript, mature 18S or 28S rRNAs, or their intermediate processed forms as measured by qPCR and Northern hybridization. Given this, the decrease in RPA194 expression or alteration of RPA135 location upon RPA12 knockdown does not impair Pol I transcription, whereas RPA135 knockdown substantially did. Therefore, the relative levels of RPA135 and RPA194 which remain in the nucleolus upon RPA12 knockdown must be sufficient for Pol I to continue at a rate equivalent to normal basal conditions.
Due to the RPA12 cleavage activity, we hypothesized that RPA12 knockdown could affect the termination of Pol I by causing the polymerase to stall without the ability to release the nascent RNA. However, based on ChIP data there are no alterations in Pol I occupancy throughout the coding region or termination sites on the rDNA template. Using NET-seq, Clarke et al. showed that deletion of RPA12.2 in yeast led to read-through capability of the polymerase enabling it to bypass T1 and T2 termination sites to ultimately engage in termination at a later promoter-proximal Reb1 binding site [41]. These findings support earlier studies by Prescott et al. which highlighted the homology between RPA12.2, Rpb9, and C11 in the termination of yeast RNA polymerases I, II and III, respectively, and showed a loss of polymerase termination in RPA12.2 deleted yeast with readthrough transcription into the spacer region [18]. It remains a possibility that the human Pol I utilizes a similar mechanism.

Conclusion
Here we showed that the transient depletion of RPA12, a core component of the Pol I enzyme complex and an essential factor for RNA cleavage, led to a decreased steady-state protein expression of the catalytic subunit RPA194. Despite this, RPA12 is nonessential for continued rRNA synthesis and chromatin engagement of the polymerase in human cancer cells. However, long-term sustained depletion of RPA12 was not achieved. This study has the limitation that it was performed in only one cancer cell line. Additional studies will be needed to investigate the implications of RPA12 knockdown and the functional impact it has on Pol I enzyme composition, transcription, and termination. As yeast RPA12.2 conveys proofreading functions for the rRNA transcript and its depletion leads to high polymerase error rates, it is plausible that long-term ablation of its activity leads to rRNA transcription errors that compromise ribosome function and cell survival.
Supporting information S1 Fig. RPA12 antibody performs poorly in detection of endogenous RPA12 in A375 melanoma cells. A375 melanoma were transfected with siRNAs against RPA12 and cell lysates were prepared after 72 hours. Cell lysates (30 μg/lane) were loaded in a 4-20% gradient gel and probed with 1:100 dilution of anti-RPA12 antibody (D10). Non-specific higher molecular weight bands were detected, whereas the detection of bands at 14 kDa expected for RPA12 was unreliable. Thereafter the membranes were probed for RPA194 (195 kDa) and histone H3 (